Continuous flow, size-based separation of entities down to the nanometer scale using nanopillar arrays

ABSTRACT

A technique relates sorting entities. The entities are introduced into a nanopillar array. The entities include a first population and a second population, and the nanopillar array includes nanopillars arranged to have a gap separating one from another. The nanopillars are ordered to have an array angle relative to a fluid flow direction. The entities are sorted through the nanopillar array by transporting the first population of the entities less than a predetermined size in a first direction and by transporting the second population of the entities at least the predetermined size in a second direction different from the first direction. The nanopillar array is configured to employ the gap with a gap size less than 300 nanometers in order to sort the entities having a sub-100 nanometer size.

DOMESTIC PRIORITY

This application claims priority to U.S. Non-Provisional ApplicationSer. No. 14/697,072, filed Apr. 27, 2015, which claims benefit to U.S.Provisional Application Ser. No. 62/084,647, filed Nov. 26, 2014, bothof which are incorporated herein by reference in their entirety.

BACKGROUND

The present invention relates to a continuous flow size-based separationof entities, and more specifically, to separating entities using ananopillar array structure.

The separation and sorting of biological entities, such as cells,proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), etc., isimportant to a vast number of biomedical applications includingdiagnostics, therapeutics, cell biology, and proteomics.

Protein and DNA/RNA separation for analytical purposes is traditionallydone by gel electrophoresis, where a protein mix is subjected to astrong electric field (typically 30 volts per centimeter (V/cm)).Proteins or DNA/RNA move through the gel at a rate depending on theirsize and surface charge. The gels are prepared from agarose oracrylamide polymers that are known to be toxic. The outcome of theelectrophoresis experiment is revealed optically from staining theproteins with dye, or staining the DNA/RNA with ethydium bromide whichis extremely carcinogenic. Gels require sufficient quantities ofmaterial for the outcome of the electrophoresis to be detectable, butbad cross-linking in the gel matrix often leads to inconclusive resultsand the complete loss of the samples. If the gel matrix size is notadapted to the sample molecule size or if the electrophoresis is left torun for too long, the sample is also lost.

For separation of macromolecules, such as DNA, RNA, proteins, and theirfragments, gel electrophoresis is widely employed. Gel electrophoresiscurrently has a market with world-wide sales greater than $1 billiondollars per year. Gel electrophoresis applied to medical diagnosticrepresents a multibillion dollar market.

In comparison with traditional techniques, silicon (Si) nanofabricationtechnology offers much more precise and accurate control innano-structural dimensions and positioning of the same, and thus canlead to reliable sorting of particles based on their sizes. To date,Si-based Lab-on-a-Chip approaches using Si pillars arrays have shownpromise. However, only sorting in the micron (10⁶ or micrometer (μm))range has been demonstrated using these techniques, which does notaccess the nanometer dimensions required for sorting DNA, proteins, etc.

SUMMARY

According to one embodiment, a method for sorting entities is provided.The entities are introduced into a nanopillar array, and the entitiesinclude a first population and a second population. The nanopillar arrayincludes nanopillars arranged to have a gap separating one from another,and the nanopillars are ordered to have an array angle relative to afluid flow direction. The entities are sorted through the nanopillararray by transporting the first population of the entities less than apredetermined size in a first direction and by transporting the secondpopulation of the entities at least the predetermined size in a seconddirection different from the first direction. The nanopillar array isconfigured to employ the gap with a gap size less than 300 nanometers inorder to sort the entities having a sub-100 nanometer size.

According to one embodiment, a method of sorting is provided. Entitiesare introduced into a nanopillar array, and the entities include a firstpopulation and a second population. The nanopillar array includesnanopillars arranged to have a gap separating one from another, and thenanopillars are ordered to have an array angle relative to a fluid flowdirection. The entities are received based on being sorted, such thatthe first population of the entities is output in a first direction andthe second population of the entities is output in a second directiondifferent from the first direction. A gap size of the gap is tuned tosort the first population in the first direction and the secondpopulation in the second direction. The gap size is tuned according toat least one of a thickness of an oxide layer disposed on the nanopillararray and/or a chemical modification to the gap.

According to one embodiment, a method of sorting is provided. Entitiesare introduced into a nanopillar array, and the entities include a firstpopulation and a second population. The nanopillar array includesnanopillars in an ordered arrangement, and the nanopillars have achemical modification. The entities are received after sorting, suchthat the first population of the entities is output in a first directionbased on the first population having an affinity to the chemicalmodification and the second population of the entities is output in asecond direction different from the first direction.

Additional features and advantages are realized through the techniquesof the present invention. Other embodiments and aspects of the inventionare described in detail herein and are considered a part of the claimedinvention. For a better understanding of the invention with theadvantages and the features, refer to the description and to thedrawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features, and advantages ofthe invention are apparent from the following detailed description takenin conjunction with the accompanying drawings in which:

FIG. 1 is a schematic of a deterministic lateral displacement (DLD)array showing definitions of the array parameters.

FIG. 2A illustrates a schematic of particle trajectories at theinterface between a neutral region and a micro fluidic metamaterialelement.

FIG. 2B illustrates the simplest metamaterial element is an asymmetricarray of posts tilted at an angle +α relative to the channel walls andbulk fluid flow.

FIG. 2C illustrates a cross-sectional SEM image showing themicrofabricated post array.

FIG. 2D illustrates equivalent microfluidic birefringence based onparticle size showing the time-trace of a 2.7-μm red fluorescenttransiting the interface and being deflected from the normal.

FIG. 3A through 3G illustrate schematics of a process flow fornanopillar array fabrication according to an embodiment, in which:

FIG. 3A illustrates a hard mask layer disposed on a substrate;

FIG. 3B illustrates disposing a resist layer on the hard mask layer;

FIG. 3C illustrates patterning the resist layer;

FIG. 3D illustrates patterning the hard mask layer;

FIG. 3E illustrates etching the substrate into the pillar array;

FIG. 3F illustrates the pillar array with the hard mask pattern removed;and

FIG. 3G illustrates disposing an oxide layer on the pillar array.

FIGS. 4A and 4B are scanning electron microscope images of the samewafer to illustrate the result of reactive ion etching before hard masksare removed according to an embodiment.

FIGS. 4C and 4D are scanning electron microscope images of a parallelprocessed wafer to illustrate the result of reactive ion etching afterhard masks are removed according to an embodiment.

FIGS. 5A and 5B are scanning electron microscope images of another waferto illustrate a fabricated nanopillar array without a thermal oxideaccording to an embodiment.

FIGS. 5C, 5D, and 5E are scanning electron microscope images of aparallel processed wafer to illustrate the impact of growing a thermaloxide on nanopillar arrays according to an embodiment.

FIGS. 6A and 6B are scanning electron microscope images of another waferto illustrate starting with a smaller gap size according to anembodiment.

FIGS. 6C and 6D are scanning electron microscope images of a parallelprocessed wafer to illustrate the oxidation process when the initial gapsize is small according to an embodiment.

FIG. 7A illustrates a general chemical schematic of chemicalmodification to a pillar array to form sorting array surfaces accordingto an embodiment.

FIG. 7B illustrates a chemical schematic for chemical modification byapplying metal to a pillar array to form sorting array surfacesaccording to an embodiment.

FIGS. 8A through 8D are cross-sectional views illustrating chemicalmodification of sorting arrays as a means of modifying the gap sizebetween pillars according to an embodiment, in which:

FIG. 8A illustrates the gap size between pillars before chemicalmodification;

FIG. 8B illustrates the reduced gap size between pillars after chemicalmodification;

FIG. 8C illustrates an enlarged view of a reactive site in FIG. 8A; and

FIG. 8D illustrates an enlarged view of the monolayer in FIG. 8B.

FIG. 9A is a top view illustrating particle flow in a chemicallymodified sorting array with particles that have no affinity for thesurface monolayer compared to particles that do have affinity for thesurface monolayer according to an embodiment.

FIG. 9B is an enlarged view of a cross-section of the nanopillar,monolayer, and particle with affinity according to an embodiment.

FIG. 10A is a cross-sectional view illustrating pillars having gapvariation according to an embodiment.

FIG. 10B is a cross-sectional view illustrating the oxidation processthat removes the gap variation according to an embodiment.

FIG. 11 is a top view illustrating a chip (fluidic device) having thepillar array according to an embodiment.

FIG. 12 is a method of providing a fluidic apparatus (e.g., chip)according to an embodiment.

FIG. 13 is a method of forming a nanopillar array according to anembodiment.

FIG. 14 is a top view of a schematic representing an arrangement of thepillars in the nanopillar array according to an embodiment.

FIG. 15 is a schematic of the chip now with two inlets and withparticles of different sizes traversing through the nanopillar arrayaccording to an embodiment.

FIG. 16A is a scanning electron microscope image of particletrajectories for 70 nanometer diameter beads according to an embodiment.

FIG. 16B is a plot of trajectory angle as a function of velocity for the70 nanometer beads according to an embodiment.

FIG. 16C is a scanning electron microscope image of particletrajectories for 50 nanometer diameter beads according to an embodiment.

FIG. 16D is a plot of trajectory angle as a function of velocity for the50 nanometer beads according to an embodiment.

FIG. 17 is a chart of example data according to an embodiment.

FIG. 18 is a method of sorting entities according to an embodiment.

FIG. 19 is a method of sorting entities according to an embodiment.

FIG. 20 is a method of sorting entities according to an embodiment.

DETAILED DESCRIPTION

Sorting in the micron (10⁶ μm) range has been demonstrated usingSi-based Lab-on-a-Chip approaches. Additional information in this regardis further discussed in a paper entitled “Hydrodynamic Metamaterials:Microfabricated Arrays To Steer, Refract, And Focus Streams OfBiomaterials” by Keith J. Morton, et al., in PNAS 2008 105 (21)7434-7438 (published ahead of print May 21, 2008), which is hereinincorporated by reference.

The paper “Hydrodynamic Metamaterials: Microfabricated Arrays To Steer,Refract, And Focus Streams Of Biomaterials” discusses that theirunderstanding of optics came from viewing light as particles that movedin straight lines and refracted into media in which the speed of lightwas material-dependent. The paper showed that objects moving through astructured, anisotropic hydrodynamic medium in laminar,high-Peclet-number flow move along trajectories that resemble light raysin optics. One example is the periodic, micro fabricated post arrayknown as the deterministic lateral displacement (DLD) array, ahigh-resolution microfluidic particle sorter. This post array isasymmetric. Each successive downstream row is shifted relative to theprevious row so that the array axis forms an angle α relative to thechannel walls and direction of fluid flow as shown in FIG. 1. Duringoperation, particles greater than some critical size are displacedlaterally at each row by a post and follow a deterministic path throughthe array in the so-called “bumping” mode. The trajectory of bumpingparticles follows the array axis angle α. Particles smaller than thecritical size follow the flow streamlines, weaving through the postarray in a periodic “zigzag” mode.

FIG. 1 is a schematic of a deterministic lateral displacement (DLD)array showing definitions of the array parameters: The posts areperiodically arranged with spacing λ, and each downstream row is offsetlaterally from the previous row by the amount δ breaking the symmetry ofthe array. This array axis forms an angle α=tan⁻¹ (δ/λ)=tan⁻¹(ϵ) withrespect to the channel walls and therefore the direction of fluid flow.Because of the array asymmetry, fluid flow in the gaps between the postsG is partitioned into 1/ϵ slots. Each of these slots repeats every 1/ϵrows so the flow through the array is on average straight. Particlestransiting the gap near a post can be displaced into an adjacentstreamline (from slot 1 to slot 2) if the particles radius is largerthan the slot width in the gap. Therefore, larger particles aredeterministically displaced at each post and migrate at an angle α tothe flow. Smaller particles simply follow the streamline paths and flowthrough the array in the direction of fluid flow.

FIG. 2A demonstrates size-based birefringence of particles flowingthrough a hydrodynamic medium of channel-spanning microfabricated posts.Two differently sized particles are normally incident on an interfacebetween a symmetric post array (left half of channel) and an asymmetricpost array (right half). Pressure-driven fluid flow through the arraysis from left to right, its overall direction determined by the largermicro fluidic channel. FIG. 2B illustrates a schematic of particletrajectories at the interface between a neutral region and amicrofluidic metamaterial element. Particles larger than a critical sizefollow the array asymmetry, whereas smaller particle follow the fluidflow. FIG. 2B illustrates the simplest metamaterial element is anasymmetric array of posts tilted at an angle +α relative to the channelwalls and bulk fluid flow. Shown is a top-view scanning electronmicrograph (SEM) of the interface between a neutral array (α=0°) and anarray with array angle α=11.3° (the gap G=4 μm and post pitch λ=11 μmare the same for both sides). FIG. 2C illustrates a cross-sectional SEMimage showing the microfabricated post array. FIG. 2D illustratesequivalent microfluidic birefringence based on particle size showing thetime-trace of a 2.7-μm red fluorescent transiting the interface andbeing deflected from the normal. Smaller 1.1-μm green beads are notdeflected at the interface.

Array elements can be tailored to direct specific particle sizes at anangle to the flow by building arrays with design parameters shown inFIG. 1, which include obstacle size D, spacing between the posts G, andpost pitch λ. Asymmetry is determined by the magnitude of the row-to-rowshift δ and is characterized by the slope ϵ=δ/λ. The final array angleis then α=tan⁻¹(ϵ). For a given array angle, the critical particle sizefor the bumping mode is determined by the ratio between the particlediameter and the post spacing or gap. This critical particle size hasbeen previously delineated for a range of array angles between 1.0° and16°. For a given gap size, the critical size of bumping is larger atsteeper angles. By using these design criteria, streams of beads, cells,and DNA have all been moved deterministically for size-based separationapplications. For the example given in FIG. 1, which has an array angleof 11.3°, gap G=4 μm, and post pitch λ=11 μm, the threshold particlesize is ≈2.4 μm. Therefore, 2.7-μm red beads travel along the array axisangle in the bumping mode, and the 1.0-μm green beads travel alongstreamlines in the zigzag mode, as shown. The array elements and anyancillary microfluidic channels and reservoirs are fabricated in siliconwafers by using standard microfabrication techniques includingphotolithography and etching. Arrays can also be molded in PDMS by usinga similarly crafted silicon master. For the silicon etch, an optimizeddeep reactive ion etch (DRIE) is used to maintain smooth, vertical sidewalls, ensuring uniform top-to-bottom spacing between posts as shown inFIG. 2C.

Unlike the state-of-the-art, embodiments are designed to createmanufacturable silicon pillar arrays with uniform gaps between thepillars (also referred to as posts) with dimensions in the sub-100nanometer (nm) regime. These pillar arrays can be used, for example, ina bumper array configuration as described above for the sorting andseparation of biological entities at these dimensions, such as DNA, RNA,exosomes, individual proteins, and protein complexes. Particularly, thepillar arrays are designed with an oxide coating, such as a SiO₂ coatingwhich can be used to “heal” variation in the gap size along the entireaxis of the pillars. Uniform gap sizes are utilized to obtain efficientsorting, e.g., to sort a 20 nm particle from a 10 nm particle. This isparticularly challenging for gaps in the sub-100 nm regime where thereis inherent variation in gap size greater than the dimensions of theparticles to be sorted, which is limited by the reactive-ion etch (RIE)process at this scale. Demonstrated sorting pillar gaps found in thestate-of-the-art have dimensions in the micron range, and therefore, thestate-of-the-art cannot sort close to this fine of a scale disclosed inembodiments. Even for a pillar array with a very small angle pitch (alsoreferred to as array angle and critical angle), e.g. 0.57 degrees, wheresorting efficiency is highest, only a particle greater that 12% of thegap will sort. Therefore, consistent gaps in the nanometer regime arerequired to sort, for example, a protein aggregate. Sorting ofindividual proteins (e.g., size range of 1-10 nm) is traditionallyperformed using ion exchange chromatography or gel electrophoresis,which are load-and-sort techniques rather than a continuous flowSi-based solution. However, the state-of-the-art technique has noexisting solution for sorting entities in 10-100 nm scale, but theembodiments provide a solution in both of these ranges (e.g., the 1-10nm range and the 10-100 nm range). Embodiments also include chemicalmodification of the pillars via attachment and/or grafting of moleculesto further decrease a given gap to a tailored size.

For ease of understanding, sub-headings may be utilized at times. Itshould be noted that the sub-headings are for explanation purposes onlyand not limitation.

Pillar Array Fabrication

FIGS. 3A through 3G illustrate schematics of a process flow fornanopillar array fabrication according to an embodiment. In FIG. 3A,process flow 301 illustrates a substrate 302. A hard mask 304 isdisposed on top of the substrate 302. The substrate 302 may be a wafer,such as, e.g., a silicon (Si) wafer. The oxide hard mask 304 may besilicon dioxide (SiO₂) that is used for etching. Although oxide is oneexample, nitride or another hard material may be utilized. The oxidehard mask 304 may be disposed by deposition and/or growth on bulksilicon (substrate 302). The thickness of the oxide hard mask 304 mayrange from tens to several hundred nanometers, depending on the etchdepth needed to create the height of the pillars and the selectivity ofthe RIE chemistry for the substrate 302 versus the hard mask material304. Other materials may be utilized for the substrate 302 and the hardmask layer 304.

In FIG. 3B, process flow 303 illustrates disposing a resist 306 on topof the oxide hard mask 304. The resist 306 may be a positive resist or anegative resist. The thickness of the resist 306 may range from 100 nm-1μm, depending on the resist 306, hard mark (304) etch selectivity, thethickness of the hard mask 304, and nanopillar gap resolution needed.For narrow sub-100 nm gaps and shallow pillar depths, a resist thicknessrange of 100-500 nm is utilized to achieve higher resolution featureswith less variability in gap size. The resist 306 may also be amulti-layer resist stack comprised of two or more layers each withdifferent etch selectivity to improve resolution.

In FIG. 3C, process flow 305 illustrates patterning the resist 306 intoa resist pattern 308. The resist pattern 308 may be defined but is notlimited to using electron-beam lithography, nanoimprint lithography,interference lithography, extreme ultraviolet lithography, and/or deepultraviolet lithography or a combination of these techniques. The resistpattern 308 is formed into resist pillars in the pattern of the futurenanopillar array. In one case, the resist pattern 308 may includemultiple patterns for different nanopillar arrays.

Process flow 307 illustrates pattern transfer from the resist pattern308 to the oxide hard mask 304 to result in the etched hard mask pattern312 in FIG. 3D. The pattern transfer to the hard mask 304 may beperformed using reactive ion etching (RIE). Process flow 307 shows theresist pattern 308 on top of the corresponding etched hard mask pattern312.

In FIG. 3E, process flow 309 illustrates patterning the nanopillars 314to be defined in the substrate 302 underneath the etched hard maskpattern 312. The nanopillars 314 may be etched using reactive ionetching. The resist pattern 308 may be removed from on top of the etchedhard mask pattern 312 before patterning the nanopillars 314 in thesubstrate 302 or after patterning the nanopillars 314. Removing theresist pattern 308 after etching the nanopillars 314 may be performed asit can serve to avoid hard mask pattern 312 erosion that can occurduring the nanopillar 314 RIE process. Hard mask erosion, in turn, maylead to pillars with a tampered (undesired) sidewall angle.

Process flow 311 illustrates removal of the hard mask pattern 312 inFIG. 3F. The hard mask pattern 312 may be removed in dilute hydrofluoric(DHF) acid, if the hard mask material is SiO₂. Process flow 311 shows ananopillar array 320 of nanopillars 314.

To further reduce the size of gaps between each of the nanopillars 314and to reduce gap variation, process flow 313 illustrates disposingoxide 316 to cover the surface of the nanopillar array 320 formed in thesubstrate 302 in FIG. 3G. In one case, thermal oxidation may be utilizedto grow silicon dioxide 316 to cover of the surface of the nanopillararray 320 in order to narrow the gaps. In another case, the oxide 316may be deposited on the nanopillar array 320 (made of silicon), forexample using atomic layer deposition.

In general, pillar arrays include a dense array of silicon pillarsdefined by RIE followed by an oxidation operation (e.g., process flow313) that serves to narrow the gaps between the pillar posts andminimize gap variation. Nanopillar array fabrication may also include anoptional chemical modification operation where further gap scaling(i.e., reduction in size) may be required. These pillar and/or gaparrays can be implemented into angled pillar designs to concentrate asample or separate a heterogeneous mixture of biological entities at thesingle molecule level, similar to work demonstrated by the paper“Hydrodynamic Metamaterials: Microfabricated Arrays To Steer, Refract,And Focus Streams Of Biomaterials” for cell or large particle sorting.The process flow for nanopillar array fabrication in FIGS. 3A and 3B canbe utilized to create arrays of nanopillars 314 shifted in any desiredgap G spacing between the nanopillars 314, desired pillar pitch λ,desired row-to-row shift δ, and desired array angle α (also referred toas the critical angle α) (as shown in FIG. 1).

Multiple nanopillar arrays 320 (e.g., 1-N, where N is the last number ofnanopillar arrays 320) may be fabricated as discussed in FIGS. 3A and 3Bon the same substrate 302. The first nanopillar arrays 320 may have afirst set of parameters (desired gap G spacing between the nanopillars314, desired pillar pitch λ, desired row-to-row shift δ, and desiredarray angle α). The second nanopillar arrays 320 may have a second setof parameters (desired gap G spacing between the nanopillars 314,desired pillar pitch λ, desired row-to-row shift δ, and desired arrayangle α), where one or more of the first set of parameters can bedifferent from the second set of parameters. The third nanopillar arrays320 may have a third set of parameters (desired gap G spacing betweenthe nanopillars 314, desired pillar pitch λ, desired row-to-row shift δ,and desired array angle α), where one or more of the first set ofparameters can be different from and/or the same as some of the secondset of parameters, and one or more of the third set of parameters can bedifferent from and/or the same as some of the first and second set ofparameters. This same analogy can apply through the last (N) nanopillararrays 320 which may have a last (N) set of parameters (desired gap Gspacing between the nanopillars 314, desired pillar pitch λ, desiredrow-to-row shift δ, and desired array angle α), where one or more of thelast set of parameters can be different from and/or the same as any oneof first, second, third, and N−1 set of parameters.

To define the pillars and gaps, a negative-tone nanoscale lithographytechnique may be better to ensure a patterned gap size less than (<) 100nm to begin with, e.g., the pillars and gaps are defined in the resistpattern 308 shown in process flow 305. Electron-beam lithography is oneoption where pillars array patterns are smaller. However, the moremanufacturable approach of nanoimprint lithography can also be appliedas well as extreme ultraviolet (EUV) and deep ultraviolet (DUV)lithography under well controlled dose conditions. To achieve a highaspect ratio pillars, the written pattern (i.e., resist pattern 308)must be transferred to the hard mask 304 (hard mask pattern 312) beforeetching the (Si) substrate 302. High aspect ratio pillars permit largerfluidic throughput and can reduce clogging issues associated withmicro/nanofluidic features. High aspect ratio pillars are therefore auseful feature to have so long as the gap size can be maintained betweenadjacent pillars. By defining the pillars in the resist pattern 308 andtransferring them to the etched hard mask pattern 312 first, the benefitof etch selectivity increases the aspect ratio while maintaining a moreconsistent gap size when the pillar array (320) etch is performed.

Some experimental data is discussed below as example implementations.The experimental data is for explanation and not limitation. In thiscase, electron-beam lithography was utilized to define the pillardimensions (e.g., resist pattern 308) in hydrogen silsesquioxane (HSQ)as part of a double layer resist stack (e.g., resist 306), which is thentransferred to a 150 nm undensified low-temperature oxide (LTO) hardmask (e.g., etched hard mask pattern 312). Densified LTO, thermal oxideand/or SiO₂/SiN/SiO₂ hard masks may also be considered. The experimentthen used a RIE-based Si etch process to define the pillars (e.g.,pillars 314) in the substrate. Further details of the RIE process arenow described.

RIE Process Details: Dry etching was carried out in an Applied MaterialsDPSII ICP etch chamber for pattern transfer to fabricate 400 nm high Sipillars from the e-beam resist pattern. First, the developed negativetone e-beam resist (HSQ) is used to etch through an organicplanarization layer (OPL) mask using a N₂/O₂/Ar/C₂H₄ chemistry at 400watts (W) source power, 100 W bias power, and 4 millitorr (mTorr)pressure at 65° C. Then, the pattern is transferred further into a SiO₂hard mask using CF₄/CHF₃ chemistry at 500 W source power, 100 W biaspower, and 30 mTorr pressure at 65° C. The carbon hard mask is thenstripped using O₂/N₂ chemistry in an Applied Materials Axiom downstreamasher at 250° C. Using the SiO₂ hard mask, Si pillars are etched to 400nm depth using the DPS II by first a CF₄/C₂H₄ breakthrough step and thenCl₂/HBr/CF₄/He/O₂/C₂H₄ main etch at 650 W source power, 85 W bias powerand 4 mTorr pressure at 65° C. It is noted that three masks wereutilized to eventually etch the pillars, and the three masks were thedeveloped HSQ e-beam resist (mask), the OPL mask, and the SiO₂ hardmask.

Gap Analysis

FIGS. 4A, 4B, 4C, and 4D are scanning electron microscope images of theresult of this RIE process for two separate instances. FIGS. 4A and 4Billustrate the pillars (e.g., pillars 314) before the hard mask (e.g.,hard mask pattern 312) is removed (such as in process flow 309), and thetops of the pillars (pillars 314 with hard mask pattern 312 on top) havea rounded shape. The 150 nm LTO (undensified) hard mask was utilizedtogether with a RIE etch to produce the pillars 314 in FIGS. 4A and 4B.FIGS. 4C and 4D illustrate pillars (e.g., pillars 314) after hard mask(e.g., hard mask pattern 312) removal by dilute hydrofluoric acidcarried out on different wafers, and the tops of the pillars 314 areflat in FIGS. 4C and 4D. In both cases, the Si pillars bow inward at thecenter due to the high density of the pillars in the array. That is, thegaps between pillars widen at the center of the pillars 314 because thediameter of the pillars are reduced at the center. The pillars have aninward-bowed shape or an hour glass shape. It is noted that pillars atthe boundaries of the array are very vertical (not shown). Thishighlights the problem of gap non-uniformity at the nanometer scalewhere approximately (˜) 100 nm gap sizes have approximately 50 nm of gapvariation from the top of a pillar to bottom (i.e., depth or height) ofthe same pillar as seen in FIGS. 4C and 4D. The close proximity ofpillars in the array as defined by the gap caused the pillars to bowinward at the center, producing gap variation that inhibits furtherscaling. This effect has been observed on gap sizes with dimensions of250 nm and below for the etch process described above (i.e., prior todisposing the oxide layer 316).

According to an embodiment, FIGS. 5A and 5B are scanning electronmicroscope images of the fabricated nanopillar array of wafer 5 withouta 50 nm thick thermal oxide. FIGS. 5C, 5D, and 5E are scanning electronmicroscope images of wafer 7 showing the impact of growing a 50 nm thickthermal oxide (e.g., oxide layer 316) on nanopillar arrays embedded inSi according to an embodiment. On the side of the pillars, there is aright wall 505 (shown in FIGS. 5A and 5C), a bottom 510 of thesubstrate, and a left wall 515 (shown in FIG. 5B).

The processing of pillars in FIGS. 5A and 5B for wafer 5 are identicalto the processing of pillars on wafer 7 in FIGS. 5C, 5D, and 5E exceptfor the final oxidation step (only performed on wafer 7 in FIGS. 5C-5E).In the case of FIG. 5B (wafer 5), there is 26 nm of variation for thegap size of approximately 186 nm while FIG. 5D (wafer 7) shows only a 13nm variation in gap size after oxidation with the gap size narrowing toapproximately 138 nm in this case. This healing effect of oxidationoccurs as a result of oxide non-uniformity on these non-planarstructures (i.e., pillars) as shown in FIG. 5E. FIG. 5E shows thatrelative to two pillars (from a side-by-side perspective in the x-axis),the gap size between those two pillars can only vary by 13 nm from topto bottom (i.e., along the vertical axis of the y-axis) because theoxide has filled in the inward-bowed shape. Using the etch processapplied to FIGS. 5A and 5B (wafer 5), uneven oxidation on pillarfeatures is found to “heal” gap variation as shown in FIGS. 5C, 5D, 5E(wafer 7) as the oxidation proceeds more rapidly at the center of thepillars (instead of at the top and bottom), and this is shown further inFIGS. 10A and 10B.

FIGS. 6A and 6B (wafer 5) illustrate starting with a smaller gap sizesuch as 80-89 nm (varies by 9 nm) in which no oxide is disposed to fillin the hour glass shape. FIGS. 6C and 6D (wafer 7) illustrate the 50 nmoxidation step applied when the original gap size is 80-89 nm (varies by9 nm). The impact of oxidation is very apparent in FIGS. 6C and 6D wherethe same 50 nm oxidation step (discussed above in FIGS. 5C, 5D, 5E)reduces the gap size from 80-89 nm to just 21-25 nm (gap variation 4 nm)with a 12:1 (depth:gap) ratio. As seen in FIGS. 6C and 6D, oxidation onsmaller starting gap sizes (e.g., such as 80-89 nm (or smaller) beforethe oxidation step to narrow the gap and remove the inward-bow) yieldsapproximately 25 nm gaps with only a few nanometers variation (4 nm)over approximately a 300 nm etch depth, where the depth to gap ratio of300:25 results in the 12:1 ratio. This small amount of gap variation(e.g., 4 nm) and process opens up the opportunity to make custom,tunable gap sizes, particularly when these nanopillars are combined withchemical modification processes. The term high aspect ratio can pertainto structures with a depth to gap ratio of greater than 4:1, which canbe difficult to achieve at this scale in a manufacturable process.

By disposing the oxide on the pillar array as discussed herein,embodiments are configured to provide a pillar array with a gap sizethat is uniform along the vertical axis (i.e., the depth) of two pillarsthat are side-by-side (e.g., the gap size between the two side-by-sidepillars varies less than 5 nm (such as by 4 nm, 3 nm, 2 nm)). Forexample, FIGS. 10A and 10B are cross-sectional views illustrating thehealing process that removes (reduces) the gap variation and creates auniform gap size in the pillar array 320 according to an embodiment. Forillustration purposes only, two pillars 314 are shown side-by-side butthe illustration applies to all of the pillars 314 in the pillar array320. The height of the pillars 314 is shown on the y-axis, and thewidth/diameter is shown on the x-axis. The z-axis represent to length ofthe array 320, and additional pillars 314 (not shown) in the array arepositioned in front of and behind the two pillars 314. FIG. 10A showstwo example pillars 314 made out of their substrate material (substrate302). The pillars 314 are bowed inward to have an hour glass shape. InFIG. 10A, two gap sizes G1 and G2 are shown but there may be additionalgap sizes between gap sizes G1 and G2. The gap size G1 is at (near) thetop and bottom of the pillars 314. The gap size G2 is at (near) thecenter of the pillars 314. The close proximity of pillars 314 as definedby the gap size G1 in the array 320 may cause the hour glass shapebecause of the dimensional constraints of the gap size G1 imposed on theimpinging flux of reactive ions during the RIE process.

FIG. 10B shows the two example pillars 314 after disposing the oxidelayer 316. Because of the non-planar architecture, nanosize of thepillars, and the tight nano-spacing between the pillars 314 in thepillar array 320, the oxide layer 316 does not distribute evenly on thepillars 314. Instead, more oxide 316 is formed more rapidly in thecenter (cavities) of the pillars 314 than at the top and bottom of thepillars 314 in the y-axis. In other words, the bowed-in centers arefilled in at a faster rate than the tops and bottoms of the pillars 314.This uneven distribution of the oxide 316 formed on the pillars 314serves to straighten the individual pillars 314 changing them from thehour glass shape to a cylinder-like shape, which in turn makes the gapsize G5 uniform between the two pillars 314 (and any other two pillars314 side-by-side in the x-axis). Accordingly, all of the gaps G(representing the general gap size of the array) are uniform throughoutthe pillar array 320.

Chemical Modification

Interaction between the particles to be sorted and the surfaces of thearray can be tailored by using chemical modification. In general, thiscan involve the attachment and/or grafting of molecules to the surfacesof the pillar array, through physical adsorption and/or formation ofchemical bonds. Also, the chemical modification of the pillar array caninclude application of a layer(s) of material such as a metal, polymer,and/or ceramic coating, as well as changes to the oxidation state of thearray surface. Surfaces (for chemical modification) can include theareas of the sorting pillars, the walls, the ceiling, and/or the floorsof the fluidic pillar array. Additionally, chemical modification can beon any surfaces present in the inlets, outlets, drive mechanisms, and/orother fluidic channels attached to the nanofluidic device (e.g., one ormore pillar arrays).

Although the chemical modification can be applied as discussed above,the better application is the chemical modification of the sortingpillars themselves, as this allows design of the interactions betweenthe particles with the sorting array surfaces.

In one example, a small organic molecule or polymer, termed a ligand,can be chemically grafted to the surface of the pillars, such as throughcondensation of chlorosilane and/or alkoxysilanes on the pillars' nativesilicon oxide as illustrated in FIG. 7A. Also, the ligand can bechemically grafted to the surface of the pillars, such as throughthiols, amines, and/or phosphines on pillars coated with a thin layer(e.g., 10 nm) of gold or silver as illustrated in FIG. 7B. The resultinglayer of ligand molecules is a single molecule thick, i.e., a monolayer.The terminal groups of the monolayer, which are in direct contact withthe fluid and particles, determine the physochemical interactions feltby the particles as they pass through the array. Changing the terminalgroup of the ligand therefore allows tailoring of the surfaceinteractions within the array.

FIGS. 7A and 7B illustrate the general chemical schematic of methods ofchemical modification of sorting array surfaces according to anembodiment. Referring to FIG. 7A, for a generic substrate (array pillar)reactive sites (X) on the surface can be used to form chemical bondsand/or physical absorption of small molecule ligands. The attachment ofligands to the surface forms a new layer, which is a single moleculethick (i.e., the monolayer). A general ligand consists of (i) a bondinggroup (Z) which interacts with the substrate reactive site (X), (ii) abackbone which consists of a number of spacer molecules (n) thatdetermine in-large the thickness of the final monolayer, and (iii) aterminal group (A) which interacts with the interface between themonolayer and the fluid/particles in the array. The terminal group (A)interacts with the particles to be sorted. Although FIG. 7A shows thebonding group Z and reactive site X, this is just one example and thechemical modification is not meant to be limited to the one type ofreaction mechanism in this example. There are two other generalmechanistic possibilities: (1) direct bond formation, i.e. the Z groupbonds to the reactive site X in a Z—X bond, and/or (2) bond formationwith elimination, i.e. the reactive group Z—W bonds to the reactive siteX—V in a Z—X bond, with the byproducts W, V eliminated. For example, thereaction of chlorosilanes R—Si—Cl with a silanol on the silica surface,H—O—Si, forms the R—Si—O—Si bond with the elimination of HCl.

Referring to FIG. 7B, monolayers can be formed on metal layers (M)pre-eposited onto the array of pillars. For example, one or more metallayers (M) can be deposited on the pillars (e.g., after the oxidizationprocess that creates the uniform gap size), such that the pillars nowhave a metal surface (M) over the substrate (and/or over the oxide layerthat fills in the inward bow). In FIG. 7B, the bonding group isidentified with ‘Q’ as opposed to ‘Z’ in FIG. 7A. Ligands (e.g., withthe bonding group (Q)) can form coordination complexes directly with themetal surface (M) of the pillar array, forming a tightly packedmonolayer.

Chemical modification can be used to tune the pillar array to sortsmaller particles by decreasing the gap size as illustrated in FIGS. 8Aand 8B. The surfaces of the sorting pillars 314 can be modified withmolecules of various length, including aliphatic or aromaticoligomers/polymers, which effectively increase the thickness of thepillars and thereby reduce the gap space between them. By selectinglonger ligands, the gap size can be made smaller and therefore theeffective cut-off particle size lowered (i.e., smaller particles can besorted). The backbone of the ligand can be selected to provide a rangeof mechanical properties between either a rigid, tight packed molecularlayer and/or a flexible, disordered layer. Ligands can include smallorganic molecules, proteins, peptides, nucleic acids, oligosaccharides,and/or synthetic polymers. In one example, pillar surfaces are modifiedwith oligomers of polyethylene glycol (PEG) through siloxane linkages.At approximately 0.36 nm per ethylene oxide residue, for a 12 residuePEG oligomer, this produces an approximately 9 nm decrease in the gapsize; for a 20 residue PEG oligomer this is approximately a 14 nmdecrease in the gap size.

FIGS. 8A through 8D illustrate schematics of chemical modification ofsorting arrays as a means of modifying the gap size between pillarsaccording to an embodiment. Referring to FIG. 8A, for pillars 314 withtheir native oxide, a grown oxide layer, and/or a deposited layer ofalternative material, e.g. metal, ceramic, polymer, there are reactivesites (X) on the surface of the pillars. The pillars 314 have an initialgap width denoted by g. There is an array floor 805 (which is the floorof the substrate 302 on which the pillars stand). FIG. 8C shows anenlarged view 820 that depicts the empty reactive site (X) in FIG. 8A.In view 820, the reactive site (X) is not attached to a ligand, but theligand is to be applied to the pillar array 320 as shown in FIG. 8B.

Referring to FIG. 8B, chemical attachment of the ligand 810 to thepillars' surfaces forms a monolayer 815 which has a thickness determinedby the properties of the ligand packing. The added thickness of themonolayer 815 reduces the gap width (from initial gap width g) to a neweffective gap width (g_(e)). Adjustment of the ligand structure, inparticular the backbone, as well as the packing and defect density ofthe monolayer 815, can tailor the thickness of the monolayer 815 andthus the tailor the effective gap (g_(e)). The effective gap (g_(e)) isthe new physical gap size experienced by the particles as they flowthrough the array 320, and is formed from the combination of thephysical barrier of the pillars plus the added steric barrier of themonolayer. The effective gap is, in general, an approximate value,dependent on the structural, mechanical, and dynamic properties of themonolayer under the operation conditions of the particle sorting. FIG.8D shows an enlarged view 820 in which the reactive sites (X) have beenattached to the ligands 810, thereby extending the diameter of thepillars 314.

Further improvement and refining of the sorting array can be introducedthrough the terminal group(s) (A) of the ligands, which can be selectedto have specific interactions with the fluid and/or particles to besorted as shown in the schematic of FIG. 9. When the particles flowthrough the pillar array 320, interactions with the terminal groups ofthe monolayer 915 leads to increased adhesion and temporary retention onthe pillar walls of pillars 314. These interactions slow down theparticle's flow, as well as causes the particles, on average, to bepositioned more at the walls of the pillars, therefore reducing theamount of the flow field it samples. As the pitch of the array isasymmetric with respect to the average fluid flow, particles (such asparticles 910) that retain and transition between pillars 314 areeffectively moved along the critical angle of the array and are sortedout. In one example, thiol terminal groups at the end of PEG-typeligands can be used to formed disulfide linkages between transitingparticles such as proteins or other molecules labeled with thiols. Incombination with a suitable catalyst agent in the fluid, when proteins(such as particles 910) flow through the array 320 they can formdisulfide bonds with the pillars 314, temporarily arresting their flow.In another example, small segments of a chemically stable nucleic acidsuch as peptide nucleic acid (PNA) can be attached to the pillar walls,to selectively delay and sort out DNA or RNA analytes through reversiblebase pairing. In another example, patches of hydrophobic ligandsembedded within hydrophilic monolayers can be introduced onto pillars,one such pair being aliphatic hydrocarbon ligands and PEG. Thehydrophobic patches can be used to interact with hydrophobic domains onproteins, to selectively sort them from solution.

FIG. 9A provides illustration of particle flow in chemically modifiedsorting array with particles 905 that have no affinity for the surfacemonolayer 915 and particles 910 which interact with the monolayer 915.Particles 905 with no affinity follow the flow lines through the array320 (i.e., exhibit a zigzag mode) and are not subject to any stronginteractions with the pillars 314. The trajectory of these particles 905is unaffected on average, and they flow without sorting in the array320. For example, the particles 905 flow into an outlet 940. However,particles 910 with a physochemical affinity, caused by molecules ontheir surface, experience interactions with the molecules of monolayer915 on the surface of the pillars 314. The interactions can temporarilybind these particles 910 to the surface of the pillars 314, and causeparticles 910 to, on average, remain closer to the pillar walls of thepillars 314. Through several sequential binding and dissociation events,the particles 910 are transferred along the direction of the pillars 314(i.e., exhibit a bump mode in the direction of the critical angle α) andare sorted by the array 320 due to chemical affinity. The particles 910are sorted into an outlet 945. FIG. 9B is an enlarged view of across-section of the nanopillar 314, monolayer 915, and particle 910with affinity according to an embodiment.

To chemically modify the pillar array 320, the ligand can be introducedthrough chemical vapor deposition (CVD) and/or wet chemistry. To applythe metal, CVD, sputtering, and/or wet chemistry may be utilized. Twodetailed examples of chemically modifying the pillars 314 by adding amonolayer discussed for explanation purposes and not limitation, and thetwo examples using wet chemistry are provided below.

For illustration purposes, an example method of modification of amicrofluidic device using poly(ethyleneoxide) (PEG) ligand modifiers isprovided below: All glassware to be exposed to chlorosilanes, is firstwashed in an isopropanol bath saturated with potassium hydroxide for atleast 24 hours, then rinsed thoroughly with deionized water and dried inan oven at 140° C. for 12 hours.

A 100 mL round bottom flask is removed from the 140° C. oven and quicklysealed with a septum. A nitrogen gas purge is set up through the septumusing needles, and the flask allowed to purge for 10 minutes. 30 mL ofanhydrous toluene is transferred into the flask via cannula. Viasyringe, 600 μL of n-octyldecyltrichlorosilane is injected to form a 49mM solution. The flask is momentarily vortexed to mix the reagentshomogenously. This forms the passivation solution. A 500 mL reactor and3-neck head are removed from the 140° C. oven and then quickly sealedtogether, with each inlet closed with a septum. A nitrogen gas purge isset up through the septum using needles, and the flask allowed to purgefor 10 minutes. Via cannula, 20 mL of the passivation solution in the100 mL flask is transferred to the reactor. The reactor is gently shakento swish the passivation solution around the walls of the reactorthoroughly. The same is done for the 100 mL flask using the remainingpassivation solution. This gentle shaking is repeated every 10-15minutes, for 1 hr. Between shaking, the glassware is allow to sit atambient temperature. This procedure is to passivate the walls of theglassware against further silizanizaiton. The passivation solution isthen poured out of the reactor, and the reactor washed sequentially, 3×each, with toluene, acetone, isopropanol and deionized water. The sameis done for the 100 mL flask. The glassware is then returned to the 140°C. oven and allowed to dry 12-14 hours.

The 100 mL round bottom flask is removed from the 140° C. oven andquickly sealed with a septum. A nitrogen gas purge is set up through theseptum using needles, and the flask allowed to purge for 30 min. 100 mLof anhydrous toluene is transferred into the flask via cannula. Viasyringe, 100 μL of 2-(methoxypoly(ethyleneoxy)₆₋₉propyl)dimethylchlorosilane is injected to form an approximately 2 mM solution.The flask is momentarily vortexed to mix the reagents homogenously. Thismodification solution is used within the day of its preparation.

Silica/silicon based microfluidic devices (chips) are cleaned for 30 minin an oxygen plasma to remove organic surface contamination. The chipsare transferred then to a 0.1M aqueous nitric acid solution for 10 minto hydrolyze any surface siloxane bonds to silanols. The chips are thenwashed sequentially, using a squeeze bottle stream, in deionized water,acetone, ethanol, and then isopropanol. The chip is then set face-up ona fresh texwipe and immediately dried off using a stream of nitrogengas, pushing solvent from the middle to outside of the chip. The chipsare then set on a custom glass holder (which sets the chipshorizontal/face-up inside the reactor, as described below).

A 500 mL reactor and 3-neck head are removed from the 140° C. oven. Astir bead along with the glass holder and chips are set into thereactor, and then quickly sealed together, with each inlet closed with aseptum. A nitrogen gas purge is set up through the septum using needles,and the reactor allowed to purge for 30 minutes.

Via a cannula, the modification solution (with the ligand) istransferred into the reaction flask until the solution level is abovethe chips. Nitrogen positive pressure is then maintained using abubbler. The reaction is allowed to run for 2 hours, at ambienttemperature, with stirring. The reactor is then opened and the chipscleaned (one-by-one) by rinsing sequentially, using a squeeze bottlestream, toluene, acetone, isopropanol, then deionized water. The chip isthen set face-up on a fresh texwipe and immediately dried off using astream of nitrogen gas, pushing solvent from the middle to outside ofthe chip. The chips are then set in a glass holding jar with a septum. Anitrogen gas purge is set up through the septum using needles, and thechips allowed to dry overnight (approximately 12-14 hours).

Use of the sub-headings is now discontinued. FIG. 11 illustrates a chip1100 (fluidic device) having the pillar array 320 according to anembodiment. The chip 1100 has an inlet 1105 to receive fluid containingthe different sized particles (i.e., biological entities) to be sorted.The inlet 1105 may be an opening or hole in the walls around thenanopillar array 320 or may span the width of the nanopillar array 320through which fluid (e.g. water, electrolyte solutions, organicsolvents, etc.) and particles (e.g., biological entities) can flow.Particles having a size greater than the critical dimension are bumped(i.e., bumped mode) through the pillar array 320 in the direction of thecritical angle, and these particles larger than the critical dimensionare collected at outlet 940. The critical dimension is the size (e.g.,diameter) of a round shaped particle and/or persistence length of chainstructure, such as DNA, that is too large to zigzag through thenanoarray 320. Particles having a size smaller than the criticaldimension zigzag (i.e., zigzag mode) through the pillar array 320 in thedirection of fluid flow, and these smaller particles are collected atthe outlet 945. The particles having the size smaller than the criticaldimension follow the direction of the fluid flow, and are sorted throughthe outlet 945. In one case, the pillars 314 may have the chemicalmodification as discussed herein, which can further reduce the gap sizeand/or sort particles having affinity to the chemical modification. Theoutlets 940 and 945 may be openings through which the sorted particlescan flow and be collected in bins after sorting.

FIG. 12 is a method 1200 of providing a fluidic apparatus 1100 (e.g.,chip 1100) is provided according to an embodiment. Reference can be madeto FIGS. 1-11 discussed above. At block 1205, the inlet 1105 isconfigured to receive a fluid. At block 1210, the outlet (e.g., outlets940, 945) is configured to exit the fluid. The nanopillar array 320 iscoupled to the inlet and the outlet, and the nanopillar array 320 isconfigured to allow the fluid to flow from the inlet to the outlet atblock 1215.

At block 1220, the nanopillar array 320 comprises nanopillars 314arranged to separate biological entities (particles) by size. At block1225, the nanopillars 314 are arranged to have a gap G separating onenanopillar 314 from another nanopillar 314, and the gap is constructedto be in a nanoscale range (e.g., sub-100 nm).

The one nanopillar is to the side of the other nanopillar, such that thegap G is in between. The gap between the one nanopillar and the othernanopillar is uniform along a vertical axis of the one nanopillar andthe other nanopillar (such as, e.g., gap G5 as shown in FIG. 10B).

The nanopillar array comprises an oxide layer 316 applied on thenanopillars, and the oxide layer 316 causes the gap to be uniform alonga vertical axis of the one nanopillar and the another nanopillar (e.g.,the gap G5 is uniform up and down the space between the two nanopillars314 in FIG. 10B).

The oxide layer 316 causes a size of the gap (e.g., gap G5) to be assmall as about 20 nanometers while the gap remains uniform along thevertical axis (e.g., y-axis in FIG. 10B). The oxide layer 316 causesunevenness in a diameter (e.g., the diameter of pillar 314 is notuniform in FIG. 10A) of the nanopillars to be uniform in FIG. 10B,resulting in the gap being uniform along the vertical axis of the onenanopillar and the other nanopillar. An increase in a thickness of theoxide layer 316 causes a decrease in a size of the gap.

In one case, the size of the gap ranges from 20-300 nm. In another case,the size of the gap may be formed to be less than 100 nm, may be lessthan 80 nm, may be less than 60 nm, may be less than 40, may be lessthan 30, may be less than 25, etc., according to the desired size of theparticles to be separated. For example, 100 nm particles can besorted/separated with 240 nm size gaps according to an embodiment.

A monolayer (e.g., the monolayer in FIGS. 7A, 7B, monolayer 815 in FIG.8B, and/or monolayer 915 in FIG. 9A) is applied to the nanopillars 314to reduce a size of the gap. The gap having a reduced size is configuredto separate smaller entities relative to when the monolayer is notapplied to the nanopillars.

FIG. 13 is a method 1300 of forming a nanopillar array 320 according toan embodiment. Reference can be made to FIGS. 1-12.

At block 1305, the hard mask layer 304 is disposed on the substrate 302.At block 1310, the resist layer 306 is patterned into a pattern (resistpattern 308) of the nanopillar array 320 in which the resist layer 306was disposed on the hard mask layer 304.

At block 1315, the resist layer (resist pattern 308) is utilized topattern the hard mask layer 304 into the pattern (hard mask pattern 312)of the nanopillar array 320, such that both the resist layer and thehard mask layer have the pattern of the nanopillar array 320.

At block 1320, the substrate 302 is patterned into the pattern of thenanopillar array 320 such that the nanopillar array 320 is now formed,wherein the resist layer (resist pattern 308) and the hard mask layer(hard mask pattern 312) are removed and wherein nanopillars 314 in thenanopillar array have a first gap size (e.g., gap size G1 and/or G2 inFIG. 10A) in a side-to-side relationship relative to each other. Atblock 1325, the first gap size is reduced to a second gap size (e.g.,gap size G5) by disposing the oxide layer 316 on the nanopillar array320.

The resist layer is patterned into the pattern (resist pattern 308) ofthe nanopillar array 320 by at least one of electron-beam lithographyand/or nanoimprint lithography or another form of lithography.

Utilizing the resist layer to pattern the hard mask layer into thepattern of the nanopillar array comprises performing reactive ionetching to etch the hard mask into the pattern (hard mask pattern 312)of the nanopillar array 320.

Patterning the substrate 302 into the pattern of the nanopillar arraysuch that the nanopillar array is formed comprises performing reactiveion etching to etch the substrate into the nanopillar array 320.

Reducing the first gap size (e.g., gap size G1 and G2) to the second gapsize (gap size G5) by disposing the oxide layer 316 on the nanopillararray 320 comprises reducing the second gap size (e.g., to less than 300nanometers, to less than 100 nanometers, etc.).

Reducing the first gap size to the second gap size by disposing theoxide layer on the nanopillar array causes each of the nanopillars tohave a uniform shape and causes the second gap size to be uniformthroughout the nanopillar array for the side-to-side relationship of thenanopillars (as shown in FIGS. 10A and 10B). Before reducing the firstgap size to the second gap size by disposing the oxide layer, thenanopillars have an inward-bowed shape at a middle of the nanopillars ata nanoscale level. Reducing the first gap size to the second gap size bydisposing the oxide layer both fills in the inward-bowed shape at themiddle and straightens the nanopillars into a cylinder-like shape.

As discussed herein, embodiments provide silicon chips with nanopillarsand nanogaps that can separate molecules and particles by size from themicron regime down to the nanometer regime. The size of two or moreentities (particles) that can be separated depends on the size of thegaps (i.e., nanogaps) between the nanopillars. The state-of-the-art hasno technologies for sorting entities by size in the 10-100 nm scale.However, embodiments described herein provide a mechanism for sortingentities within, above, and below this range (10-100 nm). For example,embodiments can sort a 30 nm particle from a 40 nm particle.Furthermore, embodiments provide continuous flow bio-separation, whichmeans that particle sorting is continuous as fluid and the entities (tobe separated) are introduced into one or more inlets of the nanopillararray 320, and the continuous flow bio-separation nanopillar array 320continuously sorts the entities without requiring any type of reset.

For example, the technology of embodiments can be used to stream asolution mix through the chip 1100, obtaining a continuous separation ofparticles within a specified size range. A heterogeneous particlesolution is introduced at the inlet of the chip 1100 and a solution flowcarries the particles through a pillar network (i.e., pillar array 320).Particles of larger sizes bounce off the nanopillars 314 according to apreset angle (i.e., critical angle α) defined by the offset δ and thepitch λ of the nanopillars 314. In this way, the trajectory of thelarger particles is directed (bump mode) toward a specific microchannelexit (e.g., outlet 940) where the separated sample can be extracted,while smaller particles will zigzag through the nanopillars 314 parallelto the direction of fluid flow where the smaller particles exit the chip1100 through a different microchannel (e.g., outlet 945).

The improvements in embodiments allow for this type of continuous flowseparation to operate at the nanometer scale, permitting efficientseparation of bio-markers, bio-molecules, sub-cellular components,exosomes, viruses, immuno-assays, drug screening, and protein aggregateson a Si chip (such as, e.g., chip 1100). Embodiments are a significantscale down from the micron scale in state-of-the-art. The improvementover the state-of-the-art was achieved through the nanofabrication ofnanopillars capable of sorting particles at the nanoscale. Embodimentsalso demonstrate that, at this new scale, a different flow regimeapplies and improves the separation method. At this scale, dead flowareas between nanopilars are proportionally significant with respect tothe nanopilar size. The presence of these dead flow areas contributes toa narrower fluidic gap between nanopillars than the physical gap (G)defined by the nanopillar wall to wall distance. This results in theability to sort a particle size smaller to what the original theorypredicts.

FIG. 14 is a top view of a schematic representing an arrangement of thepillars 314 in the nanopillar array 320 according to an embodiment. Inthis example, the pillar array 320 may be considered as multiple pillararrays. For example, the pillar array 320 includes a symmetricpart/arrangement 1405 of pillars 314 and an asymmetric part/arrangement1410 of pillars 314. The symmetric part 1405 has a critical angle thatis (virtually) 0°, while the asymmetric part 1410 has a critical angle α(defined with respect to the z-axis in FIG. 14).

In FIG. 14, the flow stream (i.e., fluid flow direction) is horizontalon the average, and the pillar rows are tilted to an angle (i.e.,forming the critical angle α) in the asymmetric part 1410 of thenanopillar array 320. At sufficiently slow flow rates of the fluid, thedistance (gap G) between the pillars 314 together with the criticalangle define the size (smaller than a critical dimension) of theparticles that are able to follow the flow direction by zig-zaggingthrough the pillars 314, and the size (equal to and/or greater than thecritical dimension) of the particles that will be displaced (bumped) bythe angle of the pillar rows. In one case, a slow flow rate maycorrespond to a flow slower than 500 μm/s.

The pillars 314 have a diameter, a pillar pitch λ, a gap (G), and arow-to-row shift (δ). The row-to-row shift (δ) is in the asymmetric part1410 because there is no row-to-row shift in the symmetric part 1405. Inthe example of FIG. 14, two example particles of different sizes aretraversing through the pillar array 320. The larger particle 1450 isdisplaced (indicated by a dash line) across the array 320 according tothe pillar angle (i.e., critical angle α), while the smaller particle1455 follows the deterministic flow (solid line) through the array 320zig-zagging through the pillars 314.

FIG. 15 is a schematic of the chip 1100 now with two inlets and with twoparticles of different sizes traversing through the pillar array 320according to an embodiment. The larger particle 1450 is displaced alongthe dashed line across the array 320 according to the pillar angle(critical angle), while the smaller particle 1455 follows thedeterministic flow through the array by zig-zagging through the pillars314. The large nanoparticle 1450 and the smaller nanoparticle 1455 exitthe array through separate microfluidic channels. For example, the largenanoparticle 1450 (e.g., equal to and/or above the critical dimension)exits the array through outlet 940, while the small nanoparticle 1455(e.g., below the critical dimension) exits the through outlet 945. Inthis example, the fluid, which may be a buffer solution, can beintroduced through the buffer in inlet 1105. The buffer solution (alsoreferred to as a pH buffer or hydrogen ion buffer) is an aqueoussolution consisting of a mixture of a weak acid and its conjugate base,or vice versa. The sample, which includes the nanoparticles 1450 and1455 to be sorted, is introduced through the same inlet 1510. Althoughonly two particles are shown, the same sorting process applies tonumerous particles having the different sizes introduced into the sampleinlet 1510.

FIGS. 16A, 16B, 16C, and 16B illustrate experimental results of passingtwo populations of nano-beads through the nanopillar array 320 accordingto an embodiment. FIGS. 16A and 16B correspond to a population of 70 nmdiameter beads, while FIGS. 16C and 16D correspond to the population of50 nm diameter beads. In FIGS. 16A and 16C the respective beads'trajectories are recorded with a video camera on a fluorescencemicroscope. In this example, the gap size (G) between the nanopillars314 is 210 nm.

FIG. 16A is the image of particle trajectories for 70 nm beads beingdisplaced in a 5.7° critical angle array (320). FIG. 16A shows that thebead trajectory of 70 nm diameter beads is angled with respect to theflow direction. The average trajectory angle observed for the 70 nmbeads is a 5.7° angle. The marked trajectories of three 70 nm particlesare shown. The angle between the flow direction (i.e., fluid flowdirection) and particle trajectory describes the degree to which theparticle is bumping in the array 320. For these 70 nm particles, a plotof trajectory angle as a function of velocity shows a positive valueclose to the critical angle in FIG. 16B. In other words, the averagetrajectory angle of the 70 nm beads plotted in FIG. 16B is approximately5.7° which is expected for the 5.7° critical angle of the array 320 inthe experiment.

FIG. 16C is the image of particle trajectories for 50 nm diameter beadssorted in the same array 320. Under the same conditions (including thesame nanopillar array 320 with the 5.7° critical angle), the 50 nm beadsare introduced in the array 320, and their trajectories are recorded inFIG. 16C. These 50 nm particles are not displaced (i.e., not in bumpmode) in the array 320, which is observed in the trajectory angle andvelocity plot in FIG. 16D. The plot in FIG. 16D shows a near-symmetricdistribution of trajectory angles around 0° (i.e., in accord with theflow axis in the array 320). Also, FIG. 16C shows that the averagetrajectory angle followed by the 50 nm beads is close to 0°.

As seen in FIGS. 16A, 16B, 16C, and 16D, the nanopillar array 320 isconfigured to sort the 50 nm beads from the 70 nm beads, by outputtingthe 50 nm beads in a first direction along the flow axis, whileoutputting the 70 nm beads in a second direction along the criticalangle of the pillar array 320.

FIG. 17 is a chart illustrating example data of the approximate gapsizes to separate a particle of one size from a particle of another sizeutilizing the nanopillar array 320 according to an embodiment. It isnoted that the example data in chart in FIG. 17 is meant forillustration purposes and not limitation. The gap sizes (G) between thenanopillars are listed horizontally, and particle diameters are listedvertically. The result of each experiment is described as either Nodisplacement, Partial displacement, or displacement 100%. Displacement100% means that the trajectory angle of the particles over the array isthe same as the set pillar critical angle, or within 15% of this angle.Partial displacement accounts for particles trajectories ranging from 15to 85% of the nanopillar critical angle. No displacement represents anyexperiment were the trajectory angle of the particles is less than 15%of the nanopillar array critical angle.

In one implementation, embodiments rely on manufacturable (silicon)pillar arrays 320 with uniform gaps between the pillars and withdimensions in the sub-100 nm regime. These arrays 320 are for thesorting and separation of biological entities at these dimensions, suchas DNA, RNA, exosomes, individual proteins, and protein complexes.Uniform gap sizes are utilized to obtain efficient sorting, e.g., tosort a 20 nm particle from a 10 nm particle according to embodiment.This is particularly challenging for gaps in the sub-100 nm regime wherethere could be inherent variations greater than the dimensions of theparticles to be sorted. This is usually caused by non-uniformnanopatterning at this scale, and feature variations in sizes and shapesdue to the reactive-ion etch (RIE) process. Demonstrated sorting pillargaps found in the state-of-the-art have dimensions in the micron rangeand therefore cannot sort even close to this fine of a scale.

Therefore, consistent gaps in the nanometer regime are required to sort,for example, a protein aggregate. Sorting of individual proteins (sizerange of 1-10 nm) is traditionally performed using ion exchangechromatography or gel electrophoresis, which are load-and-run techniquesrather than continuous flow and thus much slower. However, embodimentsprovide a continuous flow separation process and mechanism, which isconfigured to sort individual proteins (or other particles) in the rangeof 1-10 nm, without requiring ion exchange chromatography or gelelectrophoresis.

FIG. 18 is a method 1800 for sorting entities according to anembodiment. Reference can be made to FIGS. 1-17.

At block 1805, the entities are introduced into the nanopillar array320, and the entities include a first population and a secondpopulation. The nanopillar array 320 includes nanopillars 314 arrangedto have a gap separating one from another, and the nanopillars areordered to have an array angle relative to a fluid flow direction.

At block 1810, the entities are sorted through the nanopillar array 320by transporting the first population of entities less than apredetermined critical size in a first direction (e.g., toward outlet945) and by transporting the second population of entities at least thepredetermined size in a second direction (e.g., toward outlet 940)different from the first direction.

At block 1815, the nanopillar array 320 is configured to employ the gapwith a gap size less than 300 nanometers or less than 100 nanometers inorder to sort the entities having a sub-100 nanometer size.

When the entities have a nanometer size equal to or greater than 7nanometers, the nanopillar array is configured accordingly to sort theentities having the nanometer size equal to or greater than 7nanometers. When the entities have a nanometer size equal to or greaterthan 7 nanometers, the gap size is configured accordingly to sort theentities having the nanometer size equal to or greater than 7nanometers.

A lower limit of the gap size may be about 20 nanometers. A thickness ofan oxide layer 316 applied to the nanopillar array 320 causes the gapsize of the gap to be about 20 nanometers while the gap remains uniform.In other words, the gap is uniform along the vertical axis (e.g.,y-axis) between any two nanopillars 314 (i.e., no gap variation), andeach of the gaps throughout the nanopillar array 320 has the same gapsize.

The gap size of the gap is tuned to sort the first population of theentities less than the predetermined critical size in the firstdirection while sorting the second population of the entities at leastthe predetermined size in the second direction. Tuning the gap size isbased on a thickness of the oxide layer 316 applied to the nanopillararray 320. Further tuning the gap size can be based on a monolayer(e.g., without a metal applied in FIG. 7A, and/or with a metal appliedin FIG. 7B) applied to the nanopillars by chemical modification. Thechemical modification forms a monolayer (e.g., such as monolayer 815,915) on the nanopillars 314 such that the first population has anaffinity to the monolayer and the second population has no affinity tothe monolayer. Having the affinity to the monolayer directs the firstpopulation (e.g., such as entities 910) of the entities to betransported in the first direction (e.g., to outlet 945). Not having theaffinity to the monolayer allows the second population (e.g., such asentities 905) to be bumped in the second direction to outlet 940. In onecase, both entities 905 and 910 may be about the same size, and theaffinity of entities 910 causes the entities 910 to proceed toward theoutlet 945. The entities comprise at least one of bio-markers,bio-molecules, sub-cellular components, exosomes, viruses,immuno-assays, and/or protein aggregates.

Exosomes are becoming more and more important science but are too small,e.g., 30-100 nm, to be sorted by state-of-the-art arrays. Exosomes arenow believed to be present in all body fluids, and represent a new wayof thinking about cell signaling. These small extracellular vesicles arethought to play a role in a large number of biological functions. Forexample, exosomes are a messaging system and regulation system, whichmay contain and transfer DNA, RNA, protein, etc. In the nanopillar array320, the gap size can be narrowed by the oxide layer 316 to sort onesize exosome from larger size exosome, and/or to sort the smallerexosome from a different (larger) particle. Additionally, exosomes havespecial affinity (i.e., attraction) to certain ligands. For example, amonolayer 815, 915 of the lipid membrane integrating ligands, such as[6-(pyren-2-yl)octyl]silane or 3-[(8-silyloctyl)oxy]cholesterol, can beapplied to the pillars 314 to direct the exosomes in a first directionwhile directing the different particles in a second direction becausethe different particles do not have the special affinity. Therefore,even if the different particles have a same (or similar) size as theexosomes in one case, the exosomes can still be sorted because of theirspecial affinity to the certain ligands. Although certain ligands havinga special affinity to exosomes are discussed for explanation purposes,it is understood that the certain ligands having a special affinity toexosomes are not limited to these examples.

FIG. 19 is a method 1900 of sorting entities according to an embodiment.Reference can be made to FIGS. 1-18.

At block 1905, entities to be sorted are introduced into the nanopillararray 320 (e.g., via inlet 1105 and/or inlet 1510), and the entitiesinclude a first population and a second population. The nanopillar array320 includes nanopillars 314 arranged to have a gap G separating onefrom another, and the nanopillars are ordered to have an array angle(e.g., critical angle) relative to a fluid flow direction.

At block 1910, the nanopillar array 320 is configured to receive theentities at the outlet (such as the outlet 940 and/or 945 where eachoutlet may be attached/coupled to a collection tray or collection bin)based on being sorted, such that the first population of the entitiesare output in a first direction and the second population of theentities are output in a second direction different from the firstdirection;

At block 1915, a gap size of the gap G is tuned to sort the firstpopulation in the first direction and the second population in thesecond direction, and the gap size is tuned according to at least one ofa thickness of an oxide layer 316 disposed on the nanopillar array 320and/or a chemical modification (such as in FIGS. 7-9) to the gap.

When the gap size is tuned by the oxide layer 316, the oxide layer 316reduces the gap size to a first dimension. When the gap size is tuned bythe chemical modification, the chemical modification further reduces thegap size to a second dimension, and the second dimension is smaller thanthe first dimension.

The first dimension corresponds to the oxide layer 316 reducing the gapsize to about 20 nanometers while the gap remains uniform. The seconddimension corresponds to the chemical modification (e.g., attachedligand) further reducing the gap size below 20 nanometers (e.g., afterthe oxide layer 316 has been deposited). For the second dimension, thechemical modification may reduce the gap size to 18, 16, 14, 12, and/or10 nanometers. In one case, the chemical modification may reduce the gapsize to below 10 nanometers as the second dimension. In another case,the chemical modification (using longer ligands) may reduce the gap sizeto 8, 6, 4, and/or 2 nanometers as the second dimension. If desired, thechemical modification can nearly close the gap by reducing the gap sizeto less than 2 nanometers as the second dimension.

When the gap size is tuned by the chemical modification, the chemicalmodification reduces the gap size to a first dimension. It iscontemplated that the chemical modification may be applied to thenanopillars 314 even in a case when the oxide layer 316 is not applied.

The chemical modification forms a monolayer on the nanopillars such thatthe first population has an affinity to the monolayer and the secondpopulation has no affinity to the monolayer. Having the affinity to themonolayer directs the first population of the entities to be output inthe first direction. The entities comprise at least one of bio-markers,bio-molecules, sub-cellular components, exosomes, viruses,immuno-assays, and/or protein aggregates.

According to an embodiment, FIG. 20 is a method 2000 of sortingentities. Reference can be made to FIGS. 1-19.

At block 2005, entities are introduced into the nanopillar array 320,and the entities including a first population and a second population.The nanopillar array 320 includes nanopillars 314 in an orderedarrangement. The nanopillars have a chemical modification. Variousillustrations of chemical modifications have been discussed in FIGS.7-9.

At block 2010, the output (e.g., outlets 940 and 945) receives theentities after sorting, such that the first population of the entitiesare output in a first direction (e.g., outlet 945 in FIG. 9A) based onthe first population having an affinity to the chemical modification andthe second population of the entities are output in a second direction(e.g., outlet 940 in FIG. 9A) different from the first direction. Also,there may be an operator who receives the sorted entities now separatedinto/through one or more outlets (outlets 940 and 945). The operator mayutilize or attach separate collection apparatuses to separately receiveand hold the collected entities.

The second population does not have the affinity to the chemicalmodification, such as entities 905 in FIG. 9A. By the second populationnot having the affinity to the chemical modification, the secondpopulation is output in the second direction (e.g., output outlet 940).

Deposition is any process that grows, coats, or otherwise transfers amaterial onto the wafer. Available technologies include, but are notlimited to, thermal oxidation, physical vapor deposition (PVD), chemicalvapor deposition (CVD), electrochemical deposition (ECD), molecular beamepitaxy (MBE) and more recently, atomic layer deposition (ALD) amongothers.

Removal is any process that removes material from the wafer: examplesinclude etch processes (either wet or dry), and chemical-mechanicalplanarization (CMP), etc.

Patterning is the shaping or altering of deposited materials, and isgenerally referred to as lithography. For example, in conventionallithography, the wafer is coated with a chemical called a photoresist;then, a machine called a stepper focuses, aligns, and moves a mask,exposing select portions of the wafer below to short wavelength light;the exposed regions are washed away by a developer solution. Afteretching or other processing, the remaining photoresist is removed.Patterning also includes electron-beam lithography, nanoimprintlithography, and reactive ion etching.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the block may occur out of theorder noted in the figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

What is claimed is:
 1. A method for sorting entities, the methodcomprising: introducing the entities into a nanopillar array, theentities including a first population and a second population, whereinthe nanopillar array includes nanopillars arranged to have a gapseparating one from another, and wherein the nanopillars are ordered tohave an array angle relative to a fluid flow direction; sorting theentities through the nanopillar array by transporting the firstpopulation of the entities less than a predetermined size in a firstdirection and by transporting the second population of the entities atleast the predetermined size in a second direction different from thefirst direction; wherein the nanopillar array is configured to employthe gap with a gap size less than 300 nanometers in order to sort theentities having a sub-100 nanometer size; wherein a cavity in thenanopillars is filled in by an oxide layer on the nanopillars such thatthe oxide layer is configured to cause the gap size of the gap to beuniform between the nanopillars.
 2. The method of claim 1, wherein whenthe entities have a nanometer size equal to or greater than 7nanometers, the nanopillar array is configured to sort the entitieshaving the nanometer size equal to or greater than 7 nanometers.
 3. Themethod of claim 1, wherein when the entities have a nanometer size equalto or greater than 7 nanometers, the gap size is configured to sort theentities having the nanometer size equal to or greater than 7nanometers.
 4. The method of claim 1, wherein a lower limit of the gapsize is about 20 nanometers.
 5. The method of claim 4, wherein athickness of the oxide layer applied to the nanopillar array causes thegap size of the gap to be about 20 nanometers while the gap remainsuniform.
 6. The method of claim 1, wherein the gap size of the gap istuned to sort the first population of the entities less than thepredetermined size in the first direction while sorting the secondpopulation of the entities at least the predetermined size in the seconddirection; wherein tuning the gap size is based on a thickness of theoxide layer applied to the nanopillar array.
 7. The method of claim 6,wherein further tuning the gap size is based on a monolayer applied tothe nanopillars by chemical modification.
 8. The method of claim 1,wherein a chemical modification forms a monolayer on the nanopillarssuch that the first population has an affinity to the monolayer and thesecond population has no affinity to the monolayer; wherein having theaffinity to the monolayer directs the first population of the entitiesto be transported in the first direction.
 9. The method of claim 1,wherein the entities comprise at least one of bio-markers,bio-molecules, sub-cellular components, exosomes, viruses,immuno-assays, and protein aggregates.